Several methods, for example, an electroporation method and a particle gun method are known for transforming monocotyledons such as wheat, corn, and rice, which are major grain crops. However, these physical gene introduction methods have disadvantages in that a gene is introduced as multiple copies or is not inserted in an intact state, and the resulting transformed plant may often develop a malformation and sterility.
Gene introduction mediated by Agrobacterium is generally used for transformation of dicotyledons. Although it has been understood that hosts of Agrobacterium are limited only to dicotyledons and Agrobacterium have no ability to infect monocotyledons (NPL 1), some attempts have been made to transform monocotyledons through Agrobacterium. 
In 1990, there were research reports suggesting that gene introduction can also be mediated by Agrobacterium in Gramineae crops such as rice, corn, and wheat. However, these reports failed to show persuasive results because these studies had a problem in reproducibility and were also insufficient for confirmation of introduced genes (NPL 2).
Improvement in Agrobacterium Method
Recent reports involve that monocotyledons such as rice and corn can also be stably and efficiently transformed using super-binary vectors carrying parts of virulence genes of super-virulent Agrobacterium (NPLs 3 and 4). These reports state that transformation mediated by Agrobacterium has, in addition to stable and highly efficient transformation, advantages in that the resulting transformed plants have fewer mutations and that the introduced genes are low in copy number and are often in the intact states. Following the success in rice and corn, further reports were issued for Agrobacterium-mediated transformation in, for example, barley (NPL 6) and sorghum (NPL 7).
Ishida et al. (1996) (NPL 4) performed Agrobacterium-mediated transformation using a corn inbred line as a material. Furthermore, Agrobacterium-mediated transformation of corn has been reported (NPLs 8 to 10). Improvements have been attempted in the efficiency in Agrobacterium-mediated transformation of corn: for example, selection of transformed cells by means of an N6 basal medium (NPL 9), addition of AgNO3 and carbenicillin to a medium (NPLs 9 and 11), and addition of cysteine to a coculture medium (NPL 10). Ishida et al. (2003) (NPL 11) have reported that the transformation efficiency in corn is improved by selecting cocultured immature corn embryos by means of a medium containing AgNO3 and carbenicillin.
Hiei et al. (2006) (NPL 12) have reported that thermal and/or centrifugation treatment(s) of immature embryos prior to inoculation with Agrobacterium enhances the transformation efficiencies of rice and corn and also enables of transforming varieties that have not been transformed before. Hiei and Komari (2006) (NPL 13) have reported that the transformation efficiency of Indica rice is enhanced by modifying the composition and gelling agent of a coculture medium.
Thus, the modifications in medium composition and selection marker gene and the pretreatment of a plant tissue slice as a material notably enhance the efficiency in Agrobacterium-mediated transformation of rice and corn, compared to the initially reported efficiency, and such modification and pretreatment have extended the range of varieties to be applied.
Use of Immature Embryo
As materials for Agrobacterium-mediated transformation of monocotyledonous crops, immature embryos and immature embryos cultured for a short period of time are most appropriate, and immature embryos of crops such as corn, wheat, and barley are main targets of Agrobacterium infection (Cheng et al., (2004): NPL 14).
In corn and sorghum among major grain crops, immature embryos immediately after isolation are inoculated with Agrobacterium and are cocultured, and then transformed cells and transformed plants are selected (Frame et al., (2006): NPL 15, Ishida et al., (2007): NPL 16, Zhao et al., (2000): NPL 7, Gurel et al., (2009): NPL 17).
In transformation of rice using the immature embryo immediately after isolation as a material, the embryonic axis is excised from the immature embryo that has been inoculated with Agrobacterium and cocultured. The embryonic axis is removed after the coculture for removing extended bud and root (Hiei and Komari (2006): NPL 13, Datta and Datta (2006): NPL 18).
In barley, the embryonic axis is excised from the immature embryo or is wounded prior to inoculation with Agrobacterium, and then transformation mediated by Agrobacterium is performed (Tingay et al., (1997): NPL 6, Sharawat et al., (2007): NPL 19). It is difficult to remove the embryonic axis from an isolated barley immature embryo; hence constant preparation of a satisfactory immature embryo requires training for several days (Jacobsen et al., (2006): NPL 20). This troublesome process is conducted for increasing the rate of callus formation from immature embryos (Sharawat et al., (2007): NPL 19).
Thus, in the case of an immature embryo used as a material for transformation of the above-described grain crops, it is clearly determined whether or not the embryonic axis is removed from the immature embryo and when the removal is performed in the case where the embryonic axis is removed, depending on the type of the crop.
It has been also reported on attempts of producing a transformed plant of wheat, which is one of the main grain crops, mediated by Agrobacterium using the immature embryo.
For example, Cheng et al. (1997) (NPL 5) have reported that transformants can be obtained at an efficiency of 0.14% to 4.3% by inoculating immature embryos, precultured immature embryos, and calluses derived from immature embryos of wheat (variety: Bobwhite) with Agrobacterium and selecting transformed cells and plants by means of a medium containing G418. Furthermore, production of transformed wheat through Agrobacterium has been reported, but even today, more than ten years have passed from the first report by Cheng et al. (1997) (NPL 5), the efficiency is less than 5% in most reports, and the varieties to be applied are limited. Additional disadvantages, for example, low reproducibility of the published results, large variations in experimental results, and limited time for obtaining a satisfactory plant material.
Reports on wheat where immature embryos are used as a material lack in consistency; that is, many reports do not mention the process of excising embryonic axes, while some reports mention removal of embryonic axes (Przetakiewicz et al., (2004): NPL 21, Jones et al., (2005): NPL 22, Wan and Layton (2006): NPL 23, Wu et al., (2008): NPL 24, Khanna and Daggard (2003): NPL 25). Among these reports, in the reports mentioning removal of the embryonic axes in transformation of wheat, the embryonic axes are excised from the immature embryos before inoculation with Agrobacterium. 
Furthermore, regarding excision of the embryonic axis of a wheat immature embryo as a material for transformation, Jones et al. (2005) states that “precocious zygotic germination is a significant problem when immature embryo explants are used but can be suppressed by the addition of plant hormones such as dicamba, abscisic acid, or high levels of 2,4-D to the culture medium in the literature (NPL 22). Some authors specifically state that the embryonic axis was removed or damaged to prevent zygotic germination”. It is comprehended from the description above that when immature embryo is used in transformation of wheat, the problem of immature embryo germination is solved using plant hormones, and in particular, treatment of embryonic axis is unnecessary.
In addition, it is disclosed that the target tissue (embryo) of an immature wheat seed is inoculated with Agrobacterium at the time when the plant is present in its natural plant environment and is cocultured with the Agrobacterium and then that a transformed plant is obtained through dedifferentiation and regeneration of the target tissue. These literatures describe embryonic axis treatment of wheat immature embryos (PCT Japanese Translation Patent Publication No. 2002-541853 (PTL 1), Risacher et al., (2009): NPL 33). In PTL 1, the embryonic axis is removed after inoculation and coculture with Agrobacterium, but in the NPL, the embryonic axis is removed after evaluation of β-glucuronidase (GUS) expression. This means that the embryonic axis is removed after gene introduction. In addition, in the technology disclosed in PTL 1, inoculation with Agrobacterium is performed in the natural plant environment, that is, in the state where the embryo of an immature wheat seed is attached to the plant.
In NPL 33, an immature wheat seed of which target tissue (embryo) has a size of about 1 mm is inoculated with Agrobacterium by the method described in PTL 1, and the immature seed is stored as an ear for two to three days. Then, the immature embryo is isolated from the seed and is placed onto a medium containing an antibiotic for sterilizing Agrobacterium, followed by cultivation for 5 days. Subsequently, the embryonic axis is excised from the immature embryo after the cultivation, and the embryo is cultured in the same medium for 7 days, followed by selection and regeneration of the transformed cell.
As described above, in Agrobacterium-mediated transformation of wheat, transformed plants can be obtained by the known methods. However, the transformation efficiency is significantly low compared to those in rice and corn, which are the same monocotyledons as wheat, and has a problem in experimental reproducibility. Accordingly, there is a demand for developing a method capable of producing a transformant with higher efficiency and reproducibility. In addition, there are various reports on the time when the embryonic axis is removed in wheat, and a fixed knowledge has not been established yet.